Cloning, Yeast Expression, and Characterization of a β-Amyrin C-28 Oxidase (CYP716A249) Involved in Triterpenoid Biosynthesis in <i>Polygala tenuifolia</i>

Abstract

Polygala tenuifolia Willd. is a traditional Chinese herbal medicine that is widely used in treating nervous system disorders. Triterpene saponins in P. tenuifolia (polygala saponins) have excellent biological activity. As a precursor for the synthesis of presenegin, oleanolic acid (OA) plays an important role in the biosynthesis of polygala saponins. However, the mechanism behind the biosynthesis of polygala saponins remains to be elucidated. In this study, we found that CYP716A249 (GenBank: ASB17946) oxidized the C-28 position of β-amyrin to produce OA. Using quantitative real-time PCR, we observed that CYP716A249 had the highest expression in the roots of 2-year-old P. tenuifolia, which provided a basis for the selection of samples for gene cloning. To identify the function of CYP716A249, the strain R-BE-20 was constructed by expressing β-amyrin synthase in yeast. Then, CYP716A249 was co-expressed with β-amyrin synthase to construct the strain R-BPE-20 by using the lithium acetate method. Finally, we detected β-amyrin and OA by ultra-HPLC-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry and GC-MS. The results of this study provide insights into the biosynthesis pathway of polygala saponins.

The Chromosome-level genome of Aesculus wilsonii provides new insights into terpenoid biosynthesis and Aesculus evolution

“…According to the research of OSC flanking region genes in different Brassicaceae, the core genes encoding triterpenes containing OSCs, CYP450s, and ACTs to form the dynamic gene clusters, diversified the enzymes to cope with evolutionary selection pressures ( Liu et al., 2020a ). In the A. wilsonii genome, we identified a total of 320 CYP450s ( Supplementary Figure 14 ), and eight genes belonged to the CYP716 family and were reported to catalyze the C28 position in other plants ( Nelson and Werck-Reichhart, 2011 ; Zhang et al., 2020 ). In addition, we identified 153 genes from the BAHD family ( D'Auria, 2006 ; Johnson et al., 2011 ) and 46 genes from the serine carboxypeptidase-like (SCPL) acyltransferase family ( Fraser et al., 2005 ).…”

Genome assembly of Polygala tenuifolia provides insights into its karyotype evolution and triterpenoid saponin biosynthesis

“…Oxidized by cytochrome P450-dependent monooxygenases (P450s) and glycosylated by UDP-glucosyltransferases (UGTs) or cellulose synthase-like (CSLs) enzymes [ 18 ], β -amyrin finally forms different kinds of saponins [ 17 ]. Although the β AS [ 19 ] and P450 genes [ 20 ] have been identified in P. tenuifolia , other enzymes in the pathway of saponin synthesis in P. tenuifolia were not reported owing to the deficiency of genomic information.…”

Inhibitory Activity of 4-O-Benzoyl-3′-O-(OMethylsinapoyl) Sucrose from Polygala tenuifolia on Escherichia coli β-Glucuronidase

“…Polygala tenuifolia (P. tenuifolia) is an herb of the Polygalaceae family; the roots are termed "Yuan-Zhi" in Chinese [9], and have been traditionally used to enhance memory and treat cognitive dysfunctions [10]. The chemical components include saponins [11]; phenylpropanoids [12]; and xanthones [13] such as presenegenin [11], tenuifoliside A [12], and polygalaxanthone III [13]. A heteropolysaccharide in a hot water extract of P. tenuifolia reduced the expression of tumor necrosis factor-α and interleukin-6 [10].…”